Liposomes are commonly composed of phospholipid and/or sterols and consist of a vesicular structure based on lipid bilayers surrounding aqueous compartments. They vary widely in their physicochemical properties such as size, surface charge, and phospholipid composition.
Liposomes have received increasing attention as possible carriers for diagnostic or therapeutic agents. For example, liposomes have been used to deliver diagnostic agents such as contrast agents for magnetic imaging such as Gd:diethylenetriaminepentacedic acid chelate (Gd-DTPA) (See e.g. U.S. Pat. No. 6,132,763) and therapeutic agents such as anthracycline agents, which have been shown to exhibit marked activity against a wide variety of neoplasms. (See e.g. U.S. Pat. No. 4,769,250).
However, liposomes cause aggregation in the blood by their mutual reaction with various blood plasma proteins and are captured by the reticuloendothelial system (RES). For example, Kupfer cells in the liver or fixed macrophages in the spleen take up the liposomes before they can reach their intended target. Capture by the RES has rendered selected delivery of the liposomes to target tissues or cells very difficult.
In addition to capture by the RES, the liposomes are subject to electrostatic, hydrophobic, and Van der Waals interactions with plasma proteins. These interactions result in destabilization of the liposomes leading to rapid clearance of the vesicles from circulation, often before reaching their target.
Also, in addition to cellular and protein interactions with the liposomes, difficulties have arisen in producing liposome encapsulating certain drugs because of the drugs' interactions with the phospholipids of the liposomes. For example, anthracyclines have exhibited a surfactant or detergent-like effect on the phospholipid vesicle bilayer that causes leakage and creates liposome vesicle instability. Thus, liposomes unstable to the circulation environment and/or its content will leak the antineoplastic agent prematurely before reaching the tumor site. As a result of the “leaky” liposomes and the resulting devastating toxicities, scientists have tried to develop long-circulating liposomes that are able to extravasate to tumor sites, which are highly vascular in nature.
Since most commonly used anti-cancer drugs are not specifically toxic to tumor cells and are toxic to all tissues they contact, they create undesirable side effects as a result of their interactions with normal tissues. For example, Doxorubicin hydrochloride is one of the most commonly used cytotoxic anthracycline antibiotics used in cancer chemotherapy and has been shown to have activity against a wide variety of neoplasms. Doxorubicin hydrochloride is effective in the treatment of many solid tumors and leukemias. It is particularly effective in the treatment of breast cancers involving polytherapies. Doxorubicin hydrochloride is protocol therapy for AIDS related Kaposi's sarcoma. Doxorubicin hydrochloride also has notable activity against tumors of the ovaries, lung, testes, prostate, cervix, head and neck, oestrogenic sarcomas and Ewing's sarcoma.
Conventional compositions of Doxorubicin hydrochloride are available as freeze-dried product or as a solution of Doxorubicin hydrochloride in water. Freeze-dried product requires reconstitution with Water for Injection before administration. Both these marketed products have been associated with a number of toxicities when administered intravenously. Severe myelosuppression is usually the dose limiting factor. Other toxicities include nausea and vomiting, alopecia, mucositis (including stomatitis and esophagitis) and cardiotoxicity, which may limit Doxorubicin hydrochloride use. Doxorubicin hydrochloride is a potent vesicant that may cause extravasation and necrosis at the injection site or at any site that the skin is exposed. “Doxorubicin flare” is not uncommon and is characterized by erythematous streaking at the injection site. “Doxorubicin flare” usually subsides in about a half an hour.
The mechanism of action of Doxorubicin hydrochloride is not known exactly but many possibilities have been studied and described. The primary mechanism involves the ability of Doxorubicin hydrochloride to intercalate DNA. The integrity of the DNA is significantly compromised and commonly results in altered DNA functions. Single and double strand brakes are also common due to Doxorubicin hydrochloride intercalation with DNA. Another mechanism of Doxorubicin hydrochloride involves its ability to generate free radicals that induce DNA and cell membrane damage. Doxorubicin hydrochloride also inhibits topoisomerase II, rendering the reproduction of DNA ineffective.
Some of the resulting toxic affects of Doxorubicin hydrochloride include cardiac toxicity, anaphylactic reaction, emetogenicity, myelosuppression, muccocytis, skin toxicity, alopecia, and toxicity to the injection sight. (Cancer Investigation, 19 (4): 424-436 (2001)). In theory, prolonged circulation systems (slow release) that effectively deliver and release a drug to tumors and the near vicinity of tumor cells are more advantageous. Thus, it is desirable to have a stable liposome capable of encapsulating agents, such as Doxorubicin hydrochloride, that do not prematurely release their contents to healthy or non-cancerous tissues.
Several approaches taken in an effort to increase the circulation time of liposomes and thus ensure delivery of the liposome contents to the target tissue include the following: masking the liposomes from the reticuloendothelial system recognition using a sialic acid residue coating (U.S. Pat. No. 4,501,728); rigidifying the liposome membrane with sphingomyelin or neutral phospholipid with predominantly saturated acyl chains containing 5 to 20% glycolipid (U.S. Pat. No. 4,920,016); forming liposomes with a 3-80 fold higher drug to lipid ratio than traditional liposome preparations in a 3-compartment system of the agent, bilayers, and release inhibiting buffer containing citric acid (U.S. Pat. No. 6,083,530); incorporating cholesterol in the liposome (Alberto A. Gabizon, Cancer Investigation, 19(4) 424-436 (2001)); and derivatizing the phospholipid with polyethylene glycol (pegylated liposomes) (U.S. Pat. Nos. 5,013,556 and 6,132,763).
Unfortunately, the above approaches have shown only limited potential to extend the circulation time of the liposomes in vivo. For example, it has been determined that masking the liposome with sialic acid only had limited ability to extend the circulation half lives of in vivo liposomes. (U.S. Pat. No. 4,920,016). To overcome these problems, scientists have coated the liposome surface with a hydrophilic polymer such as polyethylene glycol (PEG) to prevent adsorption of various blood plasma proteins to the liposome surface. (See e.g. U.S. Pat. Nos. 5,013,556, and 5,676,971). These pegylated liposomes have been called sterically stabilized liposomes or stealth liposomes. The pegylated liposomes appeared to reduce some of the toxic effects caused by the release of their contents, but, unfortunately, new toxic effects appeared because of the presence of the polyethylene glycol. For example, the liposomal preparations containing pegylated phospholipids have lead to skin toxicity generally known as “Hand-Foot syndrome,” which results in skin eruptions/ulcers on the palms of the hands and soles of the feet. (Kenneth B. Gordon, Cancer, Vol. 75(8), 1995, 2169-2173).
Another disadvantage with pegylated liposomes is the presence of large molecules (PEG) on the liposomal surface may reduce the interactions of liposomes with cells and hinder entry of liposomes into the tumor tissue, thereby possibly reducing the accumulation of liposomal drug in the tumor tissue. (Clinical Cancer Research, (5), 1999, 3645 -3652)
Thus, there remains a need for stable, long circulating liposomes that do not cause such deleterious effects such as the “Hand-Foot syndrome” as well as methods of manufacturing such liposomes and compositions based on them. The present invention meets this need, as well as provides for methods of treatment of various conditions by administering the liposomes of the present invention.